CN103235021A - Manufacturing method and application of sensor for simultaneously detecting three breast-cancer tumor markers - Google Patents
Manufacturing method and application of sensor for simultaneously detecting three breast-cancer tumor markers Download PDFInfo
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- CN103235021A CN103235021A CN2013101367210A CN201310136721A CN103235021A CN 103235021 A CN103235021 A CN 103235021A CN 2013101367210 A CN2013101367210 A CN 2013101367210A CN 201310136721 A CN201310136721 A CN 201310136721A CN 103235021 A CN103235021 A CN 103235021A
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Abstract
The invention belongs to the sensing technical field of functional nanomaterials, clinical analysis and bioelectrochemistry, and provides a manufacturing method and an application of a sensor for simultaneously detecting three breast-cancer tumor markers. Three working electrodes are integrated on the sensor by using a printing electrode and are capable of simultaneously detecting three breast-cancer tumor markers: carcino-embryonic antigens, saccharides antigens 125 and saccharides antigens 15-3; and the detection efficiency is remarkably improved. The immunosensor has the advantages of simple establishment process and supersensitivity and has important scientific significance and application value for early clinical diagnosis of breast cancer.
Description
Technical field
The invention belongs to nano-functional material, clinical analysis and bioelectrochemical sensing technical field, a kind of preparation method and application that detects three kinds of breast cancer tumour mark sensors simultaneously is provided.
Background technology
The very high most important reason of cancer patient's mortality ratio is, can not carry out early diagnosis, if can early diagnosis in time treat, 1/3 cancer can be cured.And the mensuration of blood serum tumor markers plays an important role to the early diagnosis of cancer.Therefore, set up accurately, in the fast detecting serum tumor markers analytical approach and develop the great attention that corresponding equipment has caused people.
Breast cancer is one of modal malignant tumour of women, and according to the interrelated data statistics, the incidence of disease of breast cancer accounts for 7 ~ 10% of the various malignant tumours of whole body.Its morbidity is often relevant with heredity, it is a kind of malignant tumour that usually occurs in the breast epithelium tissue, be one of a kind of women's of having a strong impact on physical and mental health even life-threatening modal malignant tumour, and carcinomebryonic antigen (CEA), sugar antigen 125(CA125), sugar antigen 15-3(CA15-3) three kinds of tumor markerses are all closely bound up with breast cancer, and three's joint-detection has very large meaning to the early diagnosis of breast cancer.
Along with the fast development of nanometer technology in recent years, various inorganic nano materials such as carbon nano-tube, metal nanoparticle, metal oxide, semiconductor and Graphene etc. have been used for the structure of electrochemical immunosensor.
(Graphene Sheets GS) is the two-dimensional structure of a kind of individual layer, sheet to Graphene, becomes the focus that theory and practice is paid close attention in recent years.In addition, metal nano material is because its unique character and advantage, and is big as good conductivity, catalytic activity height, specific surface area, and is widely used in the enhanced sensitivity aspect of electrochemical sensor.Mesoporous nano platinum particle (M-PtNPs) wherein) has above characteristics equally, M-PtNPs self can form chemical bond-platinum ammonia key with antibody, can firmly fix antibody, and M-PtNPs has the ability of very strong catalyzing hydrogen peroxide, self can produce electric signal, be suitable for very much the preparation of sensor.
The present invention has made the triple channel screen printing electrode, and is at first grapheme modified on three working electrodes, is used for connecting and fixing the antibody of three kinds of tumor markerses.Adopt the double-antibody sandwich immunoassay method, the labeling method of two anti-(Ab2) is inquired into.Label is with M-PtNPs and Ab2, in end liquid, add hydrogen peroxide, thereby the electrochemical signals that makes immunosensor has obtained effective amplification, prepares function admirable, detects the multi-channel electrochemical immunosensor of three kinds of breast cancer tumour marks highly sensitive the time.
The present invention adopts screen printing electrode, and is cheap, and the combined with electrochemical technology makes it to have higher sensitivity; The binding immunoassay technology has improved Selectivity of Sensor; Only needing simple sample preparation, saved detection time, reduced the detection cost, is a kind of quick, cheap and sensitive detection method.
Summary of the invention
One of purpose of the present invention provides a kind of preparation method who detects three kinds of breast cancer tumour mark sensors simultaneously.
Two of purpose of the present invention is with prepared electrochemical sensor, is used for detecting simultaneously three kinds of breast cancer tumour marks.
Technical scheme of the present invention may further comprise the steps.
1. detect the preparation method of three kinds of breast cancer tumour mark sensors simultaneously, it is characterized in that, may further comprise the steps:
(1) preparation of mesoporous nano platinum particle (M-PtNPs)
K to 6 ~ 10mL, 20 mmol/L
2PtCl
4In the solution, it is 8.95 ~ 10.95 mmol/L that adding polyoxyethylene ether Brij58 makes its concentration, adds the ascorbic acid solution of 6 ~ 10 mL, 0.1 mmol/L then, ultrasonic 10 ~ 15 min of mixed solution, solution is by becoming muddy at leisure, color becomes sepia by tawny, become opaque black at last after, rotate at a high speed 35 ~ 45 min again, centrifuging, with the ultrapure water washing for several times, vacuum drying then namely obtains M-PtNPs.
(2) two anti-labels of the breast cancer tumour mark of the mesoporous nano platinum particle mark (M-PtNPs/Ab2) preparation of solution
The PBS buffer solution that the mesoporous nano platinum particle of 0.5 ~ 1.5mg is added 1mL, pH=7.0 ~ 7.8, after the ultrasonic dispersion, add a kind of in two anti-(Ab2) solution of three kinds of breast cancer tumour marks of 10 μ g, mixed solution shakes hatching 20 ~ 24h under 4 ° of C, supernatant is removed in centrifuging, and then add the PBS buffer solution of 1mL, pH=7.0 ~ 7.8, and obtain three kinds of M-PtNPs/Ab2 solution, be stored in the refrigerator of 4 ° of C standby;
The concentration of two anti-solution of described three kinds of breast cancer tumour marks is 5 ~ 10 μ g/mL.
(3) detect the preparation method of three kinds of breast cancer tumour mark sensors simultaneously
1) on the surface of 3 working electrodes of triple channel screen printing electrode, Dropwise 5 ~ 10 μ L, concentration are the Graphene solution of 2mg/mL respectively, dry naturally under the room temperature.
2) 1) in surface 1-(3-dimethylamino-propyl)-3-ethyl carbodiimide/N-hydroxy-succinamide (EDC/NHS) solution of Dropwise 5 ~ 10 μ L respectively of 2 working electrodes obtaining, keeps moistening, hatching 2 ~ 3 h, the ultrapure water cleaning is dried; The concentration of described EDC and NHS is 1mg/mL, and the mass ratio of EDC and NHS is 1 ~ 8: 1;
3) 2) in surface a kind of in the primary antibodie solution of Dropwise 5 ~ three kinds of breast cancer tumour marks of 10 μ L respectively of 3 working electrodes obtaining, place 1 ~ 2h under the room temperature, the hatching of spending the night under 4 ° of C then;
The concentration of the primary antibodie solution of described three kinds of breast cancer tumour marks is 5 ~ 10 μ g/mL.
4) there is not the primary antibodie of the described breast cancer tumour mark on crosslinked with the ultrapure water flush away, drip massfraction again and be 0.5% ~ 1% bovine serum albumin solution, under 37 ° of C, place 1h, PBS buffer solution washing with pH=7.0 ~ 7.8 is dried, under 4 ° of C, store for future use, make the sensor that detects three kinds of breast cancer tumour marks simultaneously.
2. detect three kinds of breast cancer tumour mark sensors aforesaid preparation the time, detect when being used for three kinds of breast cancer tumour marks, step is as follows:
(1) antigenic solution of three kinds of breast cancer tumour marks of concentration known is joined in the PBS buffer solution of 40 ~ 60 μ L, pH=7.0 ~ 7.8, respectively get 10 ~ 15 μ L and drip respectively on three working electrodes that are coated onto prepared sensor, place 1 h.
(2) drip respectively be coated with the mesoporous nano platinum particle mark of 5 ~ 7 μ L three kinds of M-PtNPs/Ab2 solution to corresponding working electrode, behind the 1h with the PBS buffer solution flush away of pH=7.0 ~ 7.8 not in conjunction with last label, the working electrode that obtains assembling.
(3) with contrast electrode, electrode and working electrode are connected on the electrochemical workstation, in electrolytic tank, add 40 ~ 60 μ L, 5 ~ 10 mmol/L H
2O
2The PBS buffer solution of pH=7.0 ~ 7.8, detect the current-responsive of the working electrode of assembling then with differential pulse voltammetry, according to the relation between the antigen concentration of standard solution of gained current-responsive and three kinds of breast cancer tumour marks, drawing curve.
(4) testing sample solution is replaced the standard solution of three kinds of breast cancer tumour mark antigens, detect according to the method for drafting of the working curve of the antigen of described three kinds of breast cancer tumour marks.
Three kinds of breast cancer tumour marks described in above-mentioned 1 and 2 are selected from: carcinomebryonic antigen (CEA), sugar antigen 125(CA125) and sugar antigen 15-3(CA15-3).
Described a kind of three kinds of breast cancer tumour mark sensors that detect simultaneously, used starting material all can be bought in chemical reagents corporation or biopharmaceutical company.
The present invention possesses following advantage:
(1) the present invention fast, accurately detects when can be used for three kinds of breast cancer tumour marks.
(2) mesoporous nano platinum particle and the Graphene of the present invention's employing all can strengthen electrochemical signals, thereby have improved the sensitivity of sensor of the present invention.
(3) the present invention prints electrode that preparation is simple, cost is low, is easy to carry, sample consumption is few, be easy to microminiaturized and integrated, applied range.
(4) the present invention compares with other screen printing electrode sensor, can disposablely detect three kinds of breast cancer tumour marks simultaneously, significantly improved detection efficiency, have good accuracy and precision simultaneously, the development of this immunosensor has important scientific meaning and using value to clinical early diagnosis breast cancer.
Embodiment
Further specify the present invention below in conjunction with embodiment.
Three kinds of breast cancer tumour marks described in the embodiment are selected from: carcinomebryonic antigen (CEA), sugar antigen 125(CA125) and sugar antigen 15-3(CA15-3).
Embodiment 1
The preparation of mesoporous nano platinum particle (M-PtNPs)
K to 6 mL, 20 mmol/L
2PtCl
4In the solution, it is 8.95 mmol/L that adding polyoxyethylene ether Brij58 makes its concentration, adds the ascorbic acid solution of 6 mL, 0.1 mmol/L then, ultrasonic 10 min of mixed solution, solution is by becoming muddy at leisure, its color becomes sepia by tawny, becomes opaque black at last, rotates 35 min more at a high speed, centrifuging obtains nano platinum particle, with the ultrapure water washing for several times, vacuum drying then namely obtains mesoporous nano platinum particle.
Embodiment 2
The preparation of M-PtNPs
K to 8 mL, 20 mmol/L
2PtCl
4In the solution, it is 9.95 mmol/L that adding polyoxyethylene ether Brij58 makes its concentration, adds the ascorbic acid solution of 8 mL, 0.1 mmol/L then, ultrasonic 12 min of mixed solution, solution is by becoming muddy at leisure, its color becomes sepia by tawny, becomes opaque black at last, rotates 40 min more at a high speed, centrifuging obtains nano platinum particle, with the ultrapure water washing for several times, vacuum drying then namely obtains mesoporous nano platinum particle.
Embodiment 3
The preparation of M-PtNPs
K to 10mL, 20mmol/L
2PtCl
4In the solution, it is 10.95 mmol/L that adding polyoxyethylene ether Brij58 makes its concentration, adds the ascorbic acid solution of 10 mL, 0.1mmol/L then, ultrasonic 15 min of mixed solution, solution is by becoming muddy at leisure, its color becomes sepia by tawny, becomes opaque black at last, rotates 45 min more at a high speed, centrifuging obtains nano platinum particle, with the ultrapure water washing for several times, vacuum drying then namely obtains mesoporous nano platinum particle.
Embodiment 4
Two anti-labels of the breast cancer tumour mark of the mesoporous nano platinum particle mark (M-PtNPs/Ab2) preparation of solution
The PBS buffer solution that the mesoporous nano platinum particle of 0.5mg is added 1mL, pH=7.0, after the ultrasonic dispersion, add a kind of in two anti-(Ab2) solution of three kinds of breast cancer tumour marks of 10 μ g, mixed solution shakes hatching 20h under 4 ° of C, supernatant is removed in centrifuging, and then add the PBS buffer solution of 1mL, pH=7.0, and obtain three kinds of M-PtNPs/Ab2 solution, be stored in the refrigerator of 4 ° of C standby;
The concentration of two anti-solution of described three kinds of breast cancer tumour marks is 5 μ g/mL.
Embodiment 5
The preparation of M-PtNPs/Ab2 solution
The PBS buffer solution that the mesoporous nano platinum particle of 1.5mg is added 1mL, pH=7.8, after the ultrasonic dispersion, add a kind of in two anti-(Ab2) solution of three kinds of breast cancer tumour marks of 10 μ g, mixed solution shakes hatching 24h under 4 ° of C, supernatant is removed in centrifuging, and then add the PBS buffer solution of 1mL, pH=7.8, and obtain three kinds of M-PtNPs/Ab2 solution, be stored in the refrigerator of 4 ° of C standby;
The concentration of two anti-solution of described three kinds of breast cancer tumour marks is 10 μ g/mL.
Embodiment 6
The preparation of M-PtNPs/Ab2 solution
The PBS buffer solution that the mesoporous nano platinum particle of 1.0 mg is added 1mL, pH=7.4, after the ultrasonic dispersion, add a kind of in two anti-(Ab2) solution of three kinds of breast cancer tumour marks of 10 μ g, mixed solution shakes hatching 22h under 4 ° of C, supernatant is removed in centrifuging, and then add the PBS buffer solution of 1mL, pH=7.4, and obtain three kinds of M-PtNPs/Ab2 solution, be stored in the refrigerator of 4 ° of C standby;
The concentration of two anti-solution of described three kinds of breast cancer tumour marks is 7 μ g/mL.
Embodiment 7
Detect the preparation method of three kinds of breast cancer tumour mark sensors simultaneously
(1) on the surface of 3 working electrodes of triple channel screen printing electrode, Dropwise 5 μ L, concentration are the Graphene solution of 1mg/mL respectively, dry naturally under the room temperature.
The surface of 2 working electrodes that (2) obtain in (1) is 1-(3-dimethylamino-propyl)-3-ethyl carbodiimide/N-hydroxy-succinamide (EDC/NHS) solution of Dropwise 5 μ L respectively, keeps moistening, hatching 2 h, and ultrapure water cleans, and dries; The concentration of described EDC and NHS is 1mg/mL, and the mass ratio of EDC and NHS is 1: 1;
The surface of 3 working electrodes that (3) obtain in (2) is a kind of in the primary antibodie solution of three kinds of breast cancer tumour marks of Dropwise 5 μ L respectively, places 1h under the room temperature, the hatching of spending the night under 4 ° of C then.
The concentration of the primary antibodie solution of described three kinds of breast cancer tumour marks is 5 μ g/mL.
(4) there is not the primary antibodie of the described breast cancer tumour mark on crosslinked with the ultrapure water flush away, drip massfraction again and be 0.5% bovine serum albumin solution, under 37 ° of C, place 1 h, PBS buffer solution washing with pH=7.0 is dried, under 4 ° of C, store for future use, make the sensor that detects three kinds of breast cancer tumour marks simultaneously.
Embodiment 8
Detect the preparation method of three kinds of breast cancer tumour mark sensors simultaneously
(1) on the surface of 3 working electrodes of triple channel screen printing electrode, drip 7 μ L respectively, concentration is the Graphene solution of 1.5 mg/mL, dry naturally under the room temperature.
Drip 1-(3-dimethylamino-propyl)-3-ethyl carbodiimide/N-hydroxy-succinamide (EDC/NHS) solution of 7 μ L on the surface of 2 working electrodes that (2) obtain respectively in (1), keep moistening, hatching 2.5 h, ultrapure water cleans, and dries; The concentration of described EDC and NHS is 1mg/mL, and the mass ratio of EDC and NHS is 4: 1;
Drip a kind of in the primary antibodie solution of three kinds of breast cancer tumour marks of 7 μ L on the surface of 3 working electrodes that (3) in (2), obtain respectively, place 1.5 h under the room temperature, the hatching of spending the night under 4 ° of C then.
The concentration of the primary antibodie solution of described three kinds of breast cancer tumour marks is 7 μ g/mL.
(4) there is not the primary antibodie of the described breast cancer tumour mark on crosslinked with the ultrapure water flush away, drip massfraction again and be 0.7% bovine serum albumin solution, under 37 ° of C, place 1 h, PBS buffer solution washing with pH=7.4 is dried, under 4 ° of C, store for future use, make the sensor that detects three kinds of breast cancer tumour marks simultaneously;
Embodiment 9
Detect the preparation method of three kinds of breast cancer tumour mark sensors simultaneously
(1) on the surface of 3 working electrodes of triple channel screen printing electrode, drip 10 μ L respectively, concentration is the Graphene solution of 2 mg/mL, dry naturally under the room temperature.
Drip 1-(3-dimethylamino-propyl)-3-ethyl carbodiimide/N-hydroxy-succinamide (EDC/NHS) solution of 10 μ L on the surface of 2 working electrodes that (2) obtain respectively in (1), keep moistening, hatching 3 h, ultrapure water cleans, and dries; The concentration of described EDC and NHS is 1mg/mL, and the mass ratio of EDC and NHS is 8: 1;
Drip a kind of in the primary antibodie solution of three kinds of breast cancer tumour marks of 10 μ L on the surface of 3 working electrodes that (3) in (2), obtain respectively, place 2 h under the room temperature, the hatching of spending the night under 4 ° of C then.
The concentration of the primary antibodie solution of described three kinds of breast cancer tumour marks is 10 μ g/mL.
(4) there is not the primary antibodie of the described breast cancer tumour mark on crosslinked with the ultrapure water flush away, drip massfraction again and be 1% bovine serum albumin solution, under 37 ° of C, place 1h, PBS buffer solution washing with pH=7.8 is dried, under 4 ° of C, store for future use, make the sensor that detects three kinds of breast cancer tumour marks simultaneously.
Embodiment 10
Detect in the time of three kinds of breast cancer tumour marks, step is as follows:
(1) antigenic solution of three kinds of breast cancer tumour marks of concentration known is joined in the PBS buffer solution of 40 μ L, pH=7.0, respectively get 10 μ L and drip respectively on three working electrodes that are coated onto prepared sensor, place 1 h.
(2) drip respectively be coated with the mesoporous nano platinum particle mark of 5 μ L three kinds of M-PtNPs/Ab2 solution to corresponding working electrode, behind the 1h with the PBS buffer solution flush away of pH=7.0 not in conjunction with last label, the working electrode that obtains assembling.
(3) with contrast electrode, electrode and working electrode are connected on the electrochemical workstation, in electrolytic tank, add 40 μ L, 5mmol/L H
2O
2The PBS buffer solution of pH=7.0, detect the current-responsive of the working electrode of assembling then with differential pulse voltammetry, according to the relation between the antigen concentration of standard solution of gained current-responsive and three kinds of breast cancer tumour marks, drawing curve.
(4) testing sample solution is replaced the standard solution of three kinds of breast cancer tumour mark antigens, detect according to the method for drafting of the working curve of the antigen of described three kinds of breast cancer tumour marks.
Embodiment 11
Detect in the time of three kinds of breast cancer tumour marks, step is as follows:
(1) antigenic solution of three kinds of breast cancer tumour marks of concentration known is joined in the PBS buffer solution of 60 μ L, pH=7.8, respectively get 15 μ L and drip respectively on three working electrodes that are coated onto prepared sensor, place 1 h.
(2) drip respectively be coated with the mesoporous nano platinum particle mark of 7 μ L three kinds of M-PtNPs/Ab2 solution to corresponding working electrode, behind the 1h with the PBS buffer solution flush away of pH=7.8 not in conjunction with last label, the working electrode that obtains assembling.
(3) with contrast electrode, electrode and working electrode are connected on the electrochemical workstation, in electrolytic tank, add 60 μ L, 10 mmol/L H
2O
2The PBS buffer solution of pH=7.8, detect the current-responsive of the working electrode of assembling then with differential pulse voltammetry, according to the relation between the antigen concentration of standard solution of gained current-responsive and three kinds of breast cancer tumour marks, drawing curve.
(4) testing sample solution is replaced the standard solution of three kinds of breast cancer tumour mark antigens, detect according to the method for drafting of the working curve of the antigen of described three kinds of breast cancer tumour marks.
Embodiment 12
Detect in the time of three kinds of breast cancer tumour marks, step is as follows:
(1) antigenic solution of three kinds of breast cancer tumour marks of concentration known is joined in the PBS buffer solution of 50 μ L, pH=7.4, respectively get 12 μ L and drip respectively on three working electrodes that are coated onto prepared sensor, place 1 h;
(2) drip respectively be coated with the mesoporous nano platinum particle mark of 6 μ L three kinds of M-PtNPs/Ab2 solution to corresponding working electrode, behind the 1h with the PBS buffer solution flush away of pH=7.4 not in conjunction with last label, the working electrode that obtains assembling
(3) with contrast electrode, electrode and working electrode are connected on the electrochemical workstation, in electrolytic tank, add 50 μ L, 7mmol/L H
2O
2The PBS buffer solution of pH=7.4, detect the current-responsive of the working electrode of assembling then with differential pulse voltammetry, according to the relation between the antigen concentration of standard solution of gained current-responsive and three kinds of breast cancer tumour marks, drawing curve;
(4) testing sample solution is replaced the standard solution of three kinds of breast cancer tumour mark antigens, detect according to the method for drafting of the working curve of the antigen of described three kinds of breast cancer tumour marks.
Embodiment 13
The sensor of being made by embodiment 10 ~ 12 any one methods, detect when being used for three kinds of breast cancer tumour marks of blood serum sample, adopt using standard samples recovery, every kind of parallel detection of sample 7 times, its relative standard deviation all is lower than 4.5 %, the recovery is 92.6% ~ 107%, shows that the precision of this method and accuracy are all satisfactory.
Embodiment 10 ~ 12 is used for carcinomebryonic antigen (CEA), sugar antigen 125(CA125) and sugar antigen 15-3(CA15-3) sensing range of three kinds of breast cancer tumour marks: the CEA antigen concentration is at 0.02 ~ 20 ng/mL, the CA125 antigen concentration is at 0.05 ~ 20 U/mL, the CA15-3 antigen concentration is at 0.008 ~ 24 U/mL, CEA detects and is limited to 7 pg/mL, CA125 detects and is limited to 0.002U/mL, and CA15-3 detects and is limited to 0.001 U/mL.
Claims (5)
1. detect preparation method and the application of three kinds of breast cancer tumour mark sensors simultaneously
Detect the preparation method of three kinds of breast cancer tumour mark sensors simultaneously, it is characterized in that, may further comprise the steps:
(1) preparation of mesoporous nano platinum particle (M-PtNPs)
K to 6 ~ 10mL, 20 mmol/L
2PtCl
4In the solution, it is 8.95 ~ 10.95 mmol/L that adding polyoxyethylene ether Brij58 makes its concentration, adds the ascorbic acid solution of 6 ~ 10 mL, 0.1 mmol/L then, ultrasonic 10 ~ 15 min of mixed solution, solution is by becoming muddy at leisure, color becomes sepia by tawny, become opaque black at last after, rotate at a high speed 35 ~ 45 min again, centrifuging, with the ultrapure water washing for several times, vacuum drying then namely obtains M-PtNPs;
(2) two anti-labels of the breast cancer tumour mark of the mesoporous nano platinum particle mark (M-PtNPs/Ab2) preparation of solution
The PBS buffer solution that the mesoporous nano platinum particle of 0.5 ~ 1.5mg is added 1mL, pH=7.0 ~ 7.8, after the ultrasonic dispersion, add a kind of in two anti-(Ab2) solution of three kinds of breast cancer tumour marks of 10 μ g, mixed solution shakes hatching 20 ~ 24h under 4 ° of C, supernatant is removed in centrifuging, and then add the PBS buffer solution of 1mL pH=7.0 ~ 7.8, and obtain three kinds of M-PtNPs/Ab2 solution, be stored in the refrigerator of 4 ° of C standby;
(3) detect the preparation method of three kinds of breast cancer tumour mark sensors simultaneously
1) on the surface of 3 working electrodes of triple channel screen printing electrode, Dropwise 5 ~ 10 μ L, concentration are the Graphene solution of 2 mg/mL respectively, dry naturally under the room temperature;
2) 1) in surface 1-(3-dimethylamino-propyl)-3-ethyl carbodiimide/N-hydroxy-succinamide (EDC/NHS) solution of Dropwise 5 ~ 10 μ L respectively of 2 working electrodes obtaining, keeps moistening, hatching 2 ~ 3 h, the ultrapure water cleaning is dried; The concentration of described EDC and NHS is 1mg/mL, and the mass ratio of EDC and NHS is 1 ~ 8: 1;
3) 2) in surface a kind of in the primary antibodie solution of Dropwise 5 ~ three kinds of breast cancer tumour marks of 10 μ L respectively of 3 working electrodes obtaining, place 1 ~ 2 h under the room temperature, the hatching of spending the night under 4 ° of C then;
4) there is not the primary antibodie of the described breast cancer tumour mark on crosslinked with the ultrapure water flush away, drip massfraction again and be 0.5% ~ 1% bovine serum albumin solution, under 37 ° of C, place 1 h, PBS buffer solution washing with pH=7.0 ~ 7.8 is dried, under 4 ° of C, store for future use, make the sensor that detects three kinds of breast cancer tumour marks simultaneously.
2. the preparation method who detects three kinds of breast cancer tumour mark sensors simultaneously as claimed in claim 1, the concentration of two anti-solution of three kinds of breast cancer tumour marks described in (2) is 5 ~ 10 μ g/mL.
3. the preparation method who detects three kinds of breast cancer tumour mark sensors simultaneously as claimed in claim 1 is in (3) 3) concentration of the primary antibodie solution of described three kinds of breast cancer tumour marks is 5 ~ 10 μ g/mL.
4. detect three kinds of breast cancer tumour mark sensors preparation as claimed in claim 1 the time, detect when being used for three kinds of breast cancer tumour marks, step is as follows:
(1) antigenic solution of three kinds of breast cancer tumour marks of concentration known is joined in the PBS buffer solution of 40 ~ 60 μ L, pH=7.0 ~ 7.8, respectively get 10 ~ 15 μ L and drip respectively on three working electrodes that are coated onto prepared sensor, place 1 h;
(2) drip respectively be coated with the mesoporous nano platinum particle mark of 5 ~ 7 μ L three kinds of M-PtNPs/Ab2 solution to corresponding working electrode, behind the 1h with the PBS buffer solution flush away of pH=7.0 ~ 7.8 not in conjunction with last label, the working electrode that obtains assembling;
(3) with contrast electrode, electrode and working electrode are connected on the electrochemical workstation, in electrolytic tank, add 40 ~ 60 μ L, 5 ~ 10 mmol/L H
2O
2The PBS buffer solution of pH=7.0 ~ 7.8, detect the current-responsive of the working electrode of assembling then with differential pulse voltammetry, according to the relation between the antigen concentration of standard solution of gained current-responsive and three kinds of breast cancer tumour marks, drawing curve;
(4) testing sample solution is replaced the standard solution of three kinds of breast cancer tumour mark antigens, detect according to the method for drafting of the working curve of the antigen of described three kinds of breast cancer tumour marks.
5. detect three kinds of breast cancer tumour mark sensors simultaneously as claim 1-4 is described, described three breast cancer tumour marks are selected from: carcinomebryonic antigen (CEA), sugar antigen 125(CA125) and sugar antigen 15-3(CA15-3).
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